Yeast chromatin structure and function

描述（由申请人提供）：我们的总体目标是确定染色体结构和染色质重塑酶如何影响基因组稳定性。特别是，我们对这些因子如何通过同源重组（HR）调节DNA双链断裂（DSBs）的修复以及它们如何控制复制叉的进展和稳定性感兴趣。这些途径中的任何一种缺陷都直接影响细胞存活和基因组完整性的维持，导致突变、基因易位、总体染色体重排或细胞致死。在过去的预算期间，开发了生化分析方法来剖析染色质底物上HR的早期步骤，并重建了抑制重组并要求atp依赖性染色质重塑的异染色质样结构。此外，保守的Ino80.com染色质重塑酶被证明是体内复制叉稳定性的关键调节因子。我们的总体策略是继续利用生物化学和分子遗传学方法的强大结合，以出芽酵母为实验系统，解剖dsb修复和复制过程中染色质结构的动力学。本提案中描述的实验涉及四个目标。第一个目的是研究Ino80.com染色质重塑酶在DSB加工中的作用。这个目标使用遗传方法来剖析Ino80是如何被招募到DSB的，以及它是如何促进加工的。生化研究也将描述，这将重建DSB处理在核小体底物的体外。目的2描述了在同源重组的早期步骤中，在初始连接分子形成过程中发生的染色质结构变化的生化研究。Aim 3中描述的研究将使用体内和体外方法来研究Ino80.com和Htz1组蛋白变体之间的功能相互作用。Aim 4描述了一种新的单分子、分析性超离心、组蛋白-组蛋白和组蛋白- dna交联方法的组合，以解剖Sir异染色质的结构特征。

DESCRIPTION (provided by applicant): Our overall objective is to determine how chromosome structure and chromatin remodeling enzymes influence genome stability. In particular, we are interested in how these factors regulate the repair of DNA double strand breaks (DSBs) by homologous recombination (HR) and how they control the progression and stability of replication forks. Defects in either of these pathways directly impact cell survival and maintenance of genome integrity, leading to mutations, gene translocations, gross chromosomal rearrangements, or cellular lethality. During the past budget period, biochemical assays were developed to dissect the early steps of HR on chromatin substrates, and heterochromatin-like structures were reconstituted that repress recombination and impose a requirement for ATP-dependent chromatin remodeling. In addition, the conserved Ino80.com chromatin remodeling enzyme was shown to be a key regulator of replication fork stability in vivo. Our general strategy is to continue to exploit a powerful combination of biochemical and molecular genetic approaches to dissect the dynamics of chromatin structure during the repair of DSBs and during the replication process, using budding yeast as the experimental system. Experiments described in this proposal address four aims. The first aim investigates the role of the Ino80.com chromatin remodeling enzyme in DSB processing. This aim uses genetic approaches to dissect how Ino80 is recruited to a DSB and how it contributes to processing. Biochemical studies are also described which will reconstitute DSB processing in vitro on nucleosomal substrates. Aim 2 describes biochemical studies that investigate changes in chromatin structure that occur during formation of the initial joint molecule during early steps of homologous recombination. Studies described in Aim 3 will use in vivo and in vitro methods to investigate functional interactions between Ino80.com and the Htz1 histone variant. Aim 4 describes a novel combination of single molecule, analytical ultracentrifugation, histone-histone and histone-DNA crosslinking methods to dissect the structural features of Sir heterochromatin.